Rat Diaphragm Research Articles

Pharmacodynamic studies on the isolated active fraction of Acacia farnesiana (L.) willd.

Background:Acacia farnesiana is a medicinal plant that grows throughout tropical parts of Indian subcontinent, particularly in sandy soils of river beds in Northern India. The objective of the present study was to evaluate the anti-hyperglycemic activity of the extracts using glucose tolerance test. Isolation of an active fraction (AF) from the active extract (water extract) using alcohol precipitation and to get insight to the mechanism of action of the AF of A. farnesiana.Materials and Methods:Glucose uptake by isolated rat diaphragm of the AF was performed. Further the effect of release of Insulin from isolated and cultured pancreatic β-cell was determined. Besides, effect of oral administration of the AF was compared with that of intraperitonial administration. The effect of AF on serum glucose levels in orally glucose loaded rats was compared with that of intraperitoneal glucose loaded rats.Results:The water extract significantly lowered the blood glucose level. When precipitated with alcohol, the activity was found in the soluble fraction. Glucose uptake in the isolated rat hemidiaphragm, was increased by the AF at 40 μg/ml concentration, the AF did not significantly influence insulin release from cultured islets. The AF was found to be effective in orally glucose loaded in contrast to intraperitonial route.Conclusion:Our findings suggest that this plant is promising for further studies leading to the development of valuable medicine for diabetes.

Synthesis, characterization, and preclinical evaluation of new thiazolidin-4-ones substituted with p-chlorophenoxy acetic acid and clofibric acid against insulin resistance and metabolic disorder.

We synthesized twenty thiazolidin-4-one derivatives, which were then characterized by standard chromatographic and spectroscopic methods. From the in vitro glucose uptake assay, two compounds behaved as insulin sensitizers, where they enhanced glucose uptake in isolated rat diaphragm. In high-carbohydrate diet-induced insulin resistant mice, these two thiazolidin-4-ones attenuated hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypercholesterolemia, and glucose intolerance. They raised the plasma leptin but did not reverse the diabetes-induced hypoadiponectinemia. Additionally, compound 3a reduced adiposity. The test compounds were also able to reverse the disturbed liver antioxidant milieu. To conclude, these two novel thiazolidin-4-ones modulated multiple mechanisms involved in metabolic disorders, reversing insulin resistance and thus preventing the development of type-2 diabetes.

Roles of Individual Disulfide Bridges in the Conformation and Activity of μ-Conotoxin GIIIA, a Peptide Blocker of Muscle Sodium Channels

Seven analogs of μ-conotoxin GIIIA (μ-GIIIA), a specific blocker of muscle sodium channels, were synthesized by replacing stepwise the three cystine residues with Ala. The circular dichroism spectra of the analogs suggested that the deletion of disulfide bonds gradually randamized a conformation. The inhibitory effects on the twitch contractions of the rat diaphragm showed that the deletion of one disulfide bond reduced the potency to less than 1 % of control. Monocyclic analogs and a linear analog were almost inactive. Therefore, all three disulfide bridges are essential for stabilizing the specific conformation of μ-GIIIA to show biological activity.

Molecular and physiological events in respiratory muscles and blood of rats exposed to inspiratory threshold loading

High-intensity exercise induces oxidative stress and inflammatory events in muscles. Tumor necrosis factor (TNF)-α may alter muscle protein metabolism or promote muscle regeneration. We hypothesized that a program of noninvasive chronic inspiratory loading of different intensities induces a differential pattern of physiological, molecular, and cellular events within rat diaphragms. Antioxidants and TNF-α blockade may influence those events. In the diaphragm, gastrocnemius, and blood of rats exposed to high-intensity inspiratory threshold loads (2hour every 24hours for 14days), with and without treatment with N-acetyl cysteine or infliximab (anti-TNF-α antibody), inflammatory cells and cytokines, superoxide anion production, myogenesis markers, and muscle structure were explored. In all animals, maximum inspiratory pressure (MIP) and body weight were determined. High-intensity inspiratory loading for 2 weeks caused a decline in MIP and body weight, and in the diaphragm induced a reduction in fast-twitch fiber proportions and sizes, whereas inflammatory cells and cytokine levels, including TNF-α immunohistochemical expression, superoxide anion, internal nuclei counts, and markers of myogenesis were increased. Blockade of TNF-α improved respiratory muscle function and structure, and animal weight, and, in the diaphragm, reduced inflammatory cell numbers and superoxide anion production drastically while inducing larger increases in protein and messenger RNA levels and immunohistochemical expression of TNF-α, internal nuclei, and markers of muscle regeneration. Blunting of TNF-α also induced a reduction in blood inflammatory cytokines and superoxide anion production. We conclude that TNF-α synthesized by inflammatory cells or myofibers could have differential effects on muscle structure and function in response to chronic, noninvasive, high-intensity inspiratory threshold loading.

In Memoriam: William H. Daughaday, MD (1918–2013)

Dr William H. Daughaday, the 51st President of The Endocrine Society, died at age 95 on May 3, 2013. His contributions to endocrinology and to the Society were wide-ranging and numerous, not the least of which was the large cohort of physicians attracted to fundamental and clinical endocrine research by his enthusiasm, energy, and overwhelming intellectual curiosity. Bill Daughaday grew up in greater Chicagoland and attended Harvard College. There he was captain of the wrestling team and was inducted into Phi Beta Kappa. After graduation from Harvard Medical School in 1943, where he was selected for the Alpha Omega Alpha honor society, he served in the Army as a medical officer for 20 months during the latter part of World War II and then returned stateside to continue his clinical and scientific training, initially with Robert H. Williams at Boston City Hospital. In 1947, he moved to Washington University School of Medicine in St Louis as an Assistant Resident in Medicine at Barnes Hospital. He joined the laboratory of Carl and Gerty Cori in 1949 as a Research Fellow and began to investigate the metabolic actions of GH on isolated rat diaphragm. Despite the failure of these studies to explain the (even then) well-known anti-insulin effects of GH, this apparently unpromising exploratory project set into motion a life-long interest in all things GH (1). In 1951, Bill Daughaday formed his own research laboratory and became head of a new Metabolism Division in the Department of Medicine at Washington University (he was acting head in 1950), which he continued to lead for the next 34 years. Under his guidance, the Metabolism Division developed into one of the premier endocrinology units in the country, known for high-level research productivity and superb scientific training, as evidenced by the dozens of former fellows who have occupied leadership positions in academic medicine throughout the world. This drive for excellence was amplified by combined clinical and research activities with the Division of Bone and Mineral Metabolism at neighboring Jewish Hospital of St Louis and with the Division of Endocrinology and Metabolism at St Louis Children’s Hospital. The environment nurtured by Bill guaranteed that trainees

Dexamethasone-induced hyposensitivity to rocuronium in rat diaphragm associated with muscle-fiber transformation

The aim of the current study was to investigate the effect of chronic dexamethasone (Dex) administration on rat diaphragm function and sensitivity to rocuronium and muscle‑fiber transformation. Adult male Sprague‑Dawley rats were randomized to receive a daily intraperitoneal injection of Dex to evaluate whether alterations in diaphragm function and susceptibility to rocuronium would be induced. In addition, diaphragm contractile properties, histopathology and isometric twitch tensions of nerve‑hemidiaphragm preparations were evaluated. Dex administration led to impaired diaphragm force generation, increased fatigue resistance and a prolonged half‑relaxation time, as well as time‑to‑peak tension. Dex treatment led to desensitization of the rat diaphragm to rocuronium, as demonstrated by a shift of the rocuronium concentration‑twitch tension curves to the right. Histochemical analysis of adenosine triphosphatase revealed that the distribution and cross‑sectional area of type II fibers were decreased in rats exposed to Dex. The present study indicates that chronic Dex treatment induced alterations in muscle function and that susceptibility to rocuronium is associated with muscle fiber‑type transformation, which may aid in directing future administration of muscle relaxants.

Rebuttal from Scott K. Powers, Ashley J. Smuder, David Fuller and Sanford Levine

Drs Sieck and Mantilla have presented an eloquent argument that diaphragm muscle fibre atrophy following unilateral phrenicotomy, tetrodotoxin (TTX) nerve blockade or high cervical spinal hemisection injury does not result from muscle inactivity alone. Nonetheless, we retain our position that when diaphragmatic contractile work is diminished during prolonged mechanical ventilation (MV), the ensuing diaphragm fibre atrophy occurs primarily due to decreased diaphragm contractile activity. In our response to Dr Sieck and Mantilla's position, we highlight several fundamental differences between the unilateral denervation model of diaphragm inactivity and the reduced diaphragmatic contractile work that occurs during prolonged MV. A fundamental difference between these two models of diaphragm inactivity is that prolonged MV results in atrophy of all diaphragm muscle fibre types whereas unilateral phrenic denervation promotes a transient hypertrophy of diaphragmatic type I and IIa fibres followed by atrophy of type IIx and IIb fibres. This is a unique physiological response as muscle hypertrophy does not occur in any other model of skeletal muscle inactivity. Another major difference is that diaphragm movement patterns differ markedly between the unilateral phrenic denervation model of diaphragm inactivity and MV. During MV, both hemidiaphragms move synchronously as the lungs inflate and empty. In contrast, following unilateral phrenic denervation, the contralateral hemidiaphragm contracts and shortens, thus eliciting small fibre length changes in the paralysed hemidiaphragm (Zhan et al. 1995). Since muscle fibre size is a highly regulated process controlled by mechanical signals, neurotrophic influences, and paracrine signalling which regulate muscle protein turnover (Rennie et al. 2004), the possibility exists that distortion of the paralysed diaphragm elicits an anabolic signalling response to promote hypertrophy. Importantly, not all studies report diaphragmatic hypertrophy following denervation-induced inactivity. Indeed, a study from Dr Sieck's laboratory revealed that unilateral denervation of the rat diaphragm results in a net loss of muscle protein after 5 days (Argadine et al. 2009). It follows that this deficit of muscle protein should translate into diaphragm fibre atrophy but unfortunately, this study did not report diaphragm fibre size. Further, in direct contrast to the rat data, hemidiaphragm paralysis in humans results in atrophy of diaphragm slow muscle fibres with atrophy of fast fibres occurring at a slower time course (Welvaart et al. 2011). Interestingly, these results agree with the Sieck–Mantilla initial theoretical argument that inactivity-induced diaphragm muscle fibre atrophy should occur most rapidly in slow fibres. In conclusion, based on our work and the work of others, we maintain the position that during prolonged MV, when diaphragm work against external loads is greatly diminished, the ensuing diaphragm atrophy is primarily due to decreased diaphragm contractile work.

Delivery of recombinant adeno-associated virus vectors to rat diaphragm muscle via direct intramuscular injection.

The diaphragm is the most important inspiratory muscle in all mammals, and ventilatory insufficiency caused by diaphragm dysfunction is the leading cause of morbidity and mortality in many genetic and acquired diseases affecting skeletal muscle. Currently, pharmacological inhibitors, genetically modified animals, and invasive procedures are used to study disorders affecting the diaphragm. However, these methodologies can be problematic because of off-target drug effects and the possible nonphysiological consequences of lifelong genetic alterations. Therefore, alternative methods to study this important respiratory muscle are needed. To resolve this, we have developed a methodology to deliver recombinant adeno-associated virus (rAAV) vectors to the rat diaphragm via direct intramuscular injection. We hypothesized that by direct injection of rAAV into the muscle we can selectively target the diaphragm and establish a novel experimental method for studying signaling pathways and also provide a strategy for effectively using rAAV to protect the diaphragm against disease. This report describes the methods and evidence to support the use of rAAV as a therapeutic intervention to study rat diaphragm biology during conditions that promote diaphragm dysfunction.

Chronic Actions of Nicotine on the Electrogenic Activity of Na+,K+-ATPase and the Contractile Characteristics of Rat Diaphragm

Rats received chronic nicotine via subcutaneous injections at a dose of 6 mg/kg/day for 2–3 weeks. Resting membrane potential and action potentials were recorded from muscle fibers at the end of this period, along with single and tetanic (20 and 50 impulses/sec) isometric contractions in isolated rat diaphragm. The electrogenic contribution of the α2 isoform of Na+,K+,-ATPase was assessed using ouabain (1 μM). In rats receiving chronic nicotine, the resting membrane potential was decreased by 2.2 ± 0.6 mV (p < 0.01); the electrogenic contribution of the α2 isoform of Na+,K+,-ATPase was about two times less than in the control group of animals (3.7 ± 0.6 and 6.4 ± 0.6 mV respectively, p < 0.01). Chronic nicotine had no effect on the force of single or tetanic muscle contractions in response to direct and indirect stimulation. There was a decrease in the durations of single contractions, along with a decrease in the rise time of tetanic contractions. There were no changes in the dynamics of tiring. It is suggested that chronic nicotine administration leads to decreases in the electrogenic activity of the α2 isoform of Na+,K+,-ATPase, with a resultant impairment to the electrogenesis of the diaphragm in rats.

Heparinized collagen scaffolds with and without growth factors for the repair of diaphragmatic hernia

A regenerative medicine approach to restore the morphology and function of the diaphragm in congenital diaphragmatic hernia is especially challenging because of the position and flat nature of this organ, allowing cell ingrowth primarily from the perimeter. Use of porous collagen scaffolds for the closure of surgically created diaphragmatic defects in rats has been shown feasible, but better ingrowth of cells, specifically blood vessels and muscle cells, is warranted. To stimulate this process, heparin, a glycosaminoglycan involved in growth factor binding, was covalently bound to porous collagenous scaffolds (14%), with or without vascular endothelial growth factor (VEGF; 0.4 µg/mg scaffold), hepatocyte growth factor (HGF; 0.5 µg/mg scaffold) or a combination of VEGF + HGF (0.2 + 0.5 µg/mg scaffold). All components were located primarily at the outside of scaffolds. Scaffolds were implanted in the diaphragm of rats and evaluated after 2 and 12 weeks. No herniations or eventrations were observed, and in several cases, growth factor-substituted scaffolds showed macroscopically visible blood vessels at the lung site. The addition of heparin led to an accelerated ingrowth of blood vessels at 2 weeks. In all scaffold types, giant cells and immune cells were present primarily at the liver side of the scaffold, and immune cells and individual macrophages at the lung side; these cell types decreased in number from week 2 to week 12. The addition of growth factors did not influence cellular response to the scaffolds, indicating that further optimization with respect to dosage and release profile is needed.

Diaphragm Muscle Remodeling in a Rat Model of Chronic Intermittent Hypoxia

Respiratory muscle remodeling occurs in human sleep apnea--a common respiratory disorder characterized by chronic intermittent hypoxia (CIH) due to recurrent apnea during sleep. We sought to determine if CIH causes remodeling in rat sternohyoid (upper airway dilator) and diaphragm muscles. Adult male Wistar rats were exposed to CIH (n=8), consisting of 90 sec of hypoxia (5% at the nadir; SaO₂ ~80%)/90 sec of normoxia, 8 hr per day, for 7 consecutive days. Sham animals (n=8) were exposed to alternating air/air cycles in parallel. The effect of CIH on myosin heavy-chain (MHC) isoform (1, 2a, 2x, 2b) distribution, sarcoplasmic reticulum calcium ATPase (SERCA) isoform distribution, succinate dehydrogenase activity, glycerol phosphate dehydrogenase activity, and Na⁺/K⁺ ATPase pump content was determined. Sternohyoid muscle structure was unaffected by CIH treatment. CIH did not alter oxidative/glycolytic capacity or the Na⁺/K⁺-ATPase pump content of the diaphragm. CIH significantly increased the areal density of MHC 2b fibers in the rat diaphragm, and this was associated with a shift in SERCA proteins from SERCA2 to SERCA1. We conclude that CIH causes a slow-to-fast fiber transition in the rat diaphragm after just 7 days of treatment. Respiratory muscle functional remodeling may drive aberrant functional plasticity such as decreased muscle endurance, which is a feature of human sleep apnea.

Effects of heat stress on mechanical ventilation‐induced atrophy in rat diaphragm

This study investigated the effects of heat stress on mechanical ventilation‐induced atrophy in rat diaphragm. Adult male Wistar rats (n=38) were assigned randomly to control, heat stress, mechanical ventilation (MV), or heat stress + MV groups. Heat stress consisted of 1 h in a heat chamber set at 41°C, 24 h before MV. MV was performed for 12 h under anesthesia. Immediately after MV, diaphragm was removed, frozen and stored for analysis. All procedures were approved by the animal ethics committee of Juntendo University. MV significantly increased cleaved caspase‐3 (p&lt;0.05) and reduced the mean muscle fiber cross‐sectional area (CSA, p&lt;0.05). TUNEL index was not affected by MV. Heat stress resulted in a significant induction of heat shock protein72 (p&lt;0.01), and attenuated reduction of CSA (p&lt;0.05). Cleaved caspase‐3 and TUNEL index tended to be decreased by heat stress. These results indicate that 1 h whole‐body heat stress 24 h before MV may attenuate the acute diaphragmatic atrophy via an apoptotic pathway.This work was supported by a Grant‐in‐Aid for Young Scientists (B) No. 23700781 and JSPS Fellows No. 11J11122 by JSPS.

Extraocular muscles produce more non‐mitochondrial reactive oxygen species

Extraocular muscles (EOM) are constantly active, and have a high mitochondrial content. We previously showed that EOM mitochondria are different (Invest Ophthalmol Vis Sci 50:180–6, 2009). Here, we investigated this hypothesis: EOM mitochondria produce more reactive oxygen species (ROS) than mitochondria from other muscles. First, we measured ROS production in diaphragm muscle bundles and intact EOM, excised from adult male Sprague Dawley rats, and loaded with 2’,7’‐dichlorofluorescein‐diacetate (DCFH‐DA). Next, ROS generation was measured for 60 min with Amplex Red in respiring mitochondria from rat diaphragm, gastrocnemius, and EOM. Intact EOM oxidized more DCFH: 1532 ± 407 vs. 517 ± 77 arbitrary units (AU, mean ± SD), EOM and diaphragm, respectively, n=5 muscles, p=0.04. Amplex Red oxidation was not different in mitochondria from the 3 muscles during state 3 respiration (driven by glutamate/malate), inhibition of complex I with rotenone, or succinate‐driven respiration. EOM mitochondria oxidized less Amplex Red during succinate‐driven respiration combined with complex III inhibition using antimycin: 15570±2135, 13305±1020, 9502±1024 AU, diaphragm, gastrocnemius, and EOM respectively; p=0.0009, n=4 rats). These results show that intact EOM generated ROS faster than diaphragm bundles. However, EOM mitochondria did not produce more ROS than mitochondria from other muscles. Therefore, we reject our initial hypothesis, and propose that the higher rate of ROS production in EOM originates from non‐mitochondrial sources.*Research supported by APS Undergraduate Research Fellowship 2012

Mechanical ventilation impairs sarcomeric protein function in rat diaphragm single fibers

Mechanical ventilation (MV) is a widely used clinical intervention for patients unable to maintain alveolar ventilation. Although a life‐saving measure for patients suffering from respiratory failure, MV promotes diaphragm atrophy and contractile dysfunction. The cellular basis of contractile dysfunction remains unclear. The mechanisms of contractile dysfunction are important to understand because MV‐induced diaphragm weakness can delay the weaning process. We studied triton‐permeabilized diaphragm single fibers from rats after 12 hrs of spontaneous breathing (SB) and MV (n = 4/group). Key parameters of isometric contractile properties were maximal specific tetanic force (sPo), rate constant of tension redevelopment (ktr), and calcium sensitivity (pCa50). In type I diaphragm fibers, sPo did not differ between groups. MV slowed ktr by ~6% and decreased the pCa50 such that calcium concentration eliciting 50% sPo increased by ~20%. In type II diaphragm fibers, MV diminished sPo by ~32% and slowed ktr by ~22%. However, pCa50 did not differ between groups. Together, our data suggest that MV impairs diaphragm sarcomeric protein function as evidenced by diminished specific force and calcium sensitivity, and slowing of cross bridge cycling kinetics in membrane‐permeabilized single fibers.Funding support: NIH R00‐HL098453 (LFF) and HL087839 (SKP)

Impact of prolonged mechanical ventilation on diaphragmatic protein synthesis

Mechanical ventilation (MV) is a life‐saving intervention in patients suffering from respiratory failure. Unfortunately, prolonged MV promotes the rapid development of diaphragmatic atrophy due to increased proteolysis and decreased protein synthesis. These experiments investigated the influence of two different modes of MV on rat diaphragm protein synthesis. Specifically, we examined the mixed protein synthesis rate of diaphragm muscle proteins during controlled MV (CMV) which causes complete diaphragmatic inactivity and pressure support MV (PSV) which permits partial activity of the diaphragm. Our results reveal that compared to spontaneously breathing animals, diaphragm protein synthesis rates are not depressed during the first 6‐hours of PSV but are significantly decreased in animals exposed to CMV. However, the rates of diaphragm protein synthesis are depressed at similar levels in both CMV and PSV animals during 6–12 hours of MV. We conclude that while PSV can initially delay the MV‐induced decreases in diaphragm protein synthesis, this protective effect is rapidly lost after the first 6 hours of MV. NIH RO1 HL087839 (SKP)

Antidiabetic Properties and Mechanism of Action of Orthosiphon stamineus Benth Bioactive Sub-fraction in Streptozotocin-induced Diabetic Rats

Orthosiphon stamineus is a popular folk medicine widely used to treat many diseases including diabetes. Previous studies have shown that the sub-fraction of chloroform extract was able to inhibit the rise of blood glucose levels in a glucose tolerance test. This study was carried out to evaluate the chronic effect and possible mechanism of action of the bioactive chloroform sub-fraction of O. stamineus using streptozotocin-induced diabetic rats and in vitro methods. Administration of the chloroform extract sub-fraction 2 (Cƒ2-b) at a dose of 1 g/kg twice daily on diabetic rats for 14 days showed a significant lowering (p < 0.05) of the final blood glucose level compared to the pretreatment level. However, there were no significant differences in the plasma insulin levels post-treatment compared to the pretreatment levels for all doses of Cƒ2-b. Conversely, Cƒ2-b at a concentration of 2 mg/mL significantly increased (p < 0.001) the glucose uptake by the rat diaphragm muscle. The increase in glucose uptake was also shown when the muscle was incubated in a solution containing 1 IU/mL of insulin or 1 mg/mL of metformin. Furthermore, the effect of this sub-fraction on glucose absorption in the everted rat jejunum showed that Cƒ2-b at concentrations of 0.5 mg/mL, 1 mg/mL and, 2 mg/mL significantly reduced the glucose absorption of the jejunum (p < 0.05–0.001). Similarly, the absorption of glucose was also inhibited by 1 mg/mL and 2 mg/mL of metformin (p < 0.001). These results suggest that the effect of Cƒ2-b may be due to extra-pancreatic mechanisms. There was no evidence that the plant extract stimulated the release of insulin in order to lower the blood glucose level.

The Effect of Nitric Oxide on the Rat Diaphragm at Different Frequencies of Indirect Electrical Stimulation

Introduction: Nitric oxide (NO) is a second messenger that regulates neurotransmitter release at the neuromuscular junction (nmj). Evidences accumulate on its potency as a neuro-modulator in CNS, PNS. This work investigates the effect of NO on (nmj) by adding L-arginine known as NO donor. Material and Methods: 30 albino rats’ diaphragms divided into two groups. Both exposed to L-argnine followed by Bovine Hb and stimulated by indirect electrical stimulation. Gp 1 stimulated at 0.5Hz. Gp2 stimulated at 100Hz. The amplitude of maximum contraction (AMC) (ΔY), contraction time (ΔX) and half relaxation time (1⁄2 Rt) measured. Results: In presence of NO Gp1 showed a significant increase in (AMC) (ΔY) and (ΔX) with a significant decrease in (1⁄2 Rt). Gp2 showed a significant increase in (AMC)(ΔY). Conclusion: Nitric oxide (NO) facilitates neuromuscular transmission at presynaptic level. Bovine Hemoglobin reduces Nitric oxide’s induced effects.

Age and activity-related changes in the respiratory motor system

The ability of the respiratory motor system to adapt to various functional demands is very important for maintaining human lifestyles. Age-related changes in the system have been studied in several animals. Furthermore, many kinds of models of altered use have been developed to examine activity-related changes in the system. In this review, the plasticity of the respiratory motor system, including spinal phrenic motoneurons and endplates on muscle fibers are discussed, mainly relating to inactivity and over-activity models in rat diaphragm muscle.

The evaluation of the reactivating and therapeutic efficacy of three novel bispyridinium oximes (K454, K456, K458) in comparison with the oxime K203 and trimedoxime in tabun-poisoned rats and mice

The potency of three newly developed bispyridinium compounds (K454, K456, K458) to reactivate tabun-inhibited acetylcholinesterase and reduce tabun-induced lethal toxic effects was compared with the oxime K203 and trimedoxime using in vivo methods. The study determining percentage of reactivation of tabun-inhibited diaphragm and brain acetylcholinesterase in poisoned rats showed that the reactivating efficacy of all newly developed oximes is comparable with K203 but lower than the reactivating potency of trimedoxime in diaphragm. In the brain, their potency to reactivate tabun-inhibited acetylcholinesterase is lower compared with trimedoxime and the oxime K203. All three newly developed oximes were also found to be relatively effective in reducing lethal toxic effects in tabun-poisoned mice. Their therapeutic efficacy is consistent with the therapeutic potency of the oxime K203. On the other hand, their potency to reduce acute toxicity of tabun is significantly lower compared with trimedoxime. In conclusion, the reactivating and therapeutic potency of all three newly developed oximes does not prevail the effectiveness of the oxime K203 and trimedoxime and, therefore, they are not suitable for their replacement of commonly used oximes for the treatment of acute tabun poisoning.

Mechanical ventilation reduces rat diaphragm blood flow and impairs oxygen delivery and uptake*

Although mechanical ventilation is a life-saving intervention in patients suffering from respiratory failure, prolonged mechanical ventilation is often associated with numerous complications including problematic weaning. In contracting skeletal muscle, inadequate oxygen supply can limit oxidative phosphorylation resulting in muscular fatigue. However, whether prolonged mechanical ventilation results in decreased diaphragmatic blood flow and induces an oxygen supply-demand imbalance in the diaphragm remains unknown. We tested the hypothesis that prolonged controlled mechanical ventilation results in a time-dependent reduction in rat diaphragmatic blood flow and microvascular PO2 and that prolonged mechanical ventilation would diminish the diaphragm's ability to increase blood flow in response to muscular contractions. Compared to 30 mins of mechanical ventilation, 6 hrs of mechanical ventilation resulted in a 75% reduction in diaphragm blood flow (via radiolabeled microspheres), which did not occur in the intercostal muscle or high-oxidative hindlimb muscle (e.g., soleus). There was also a time-dependent decline in diaphragm microvascular PO2 (via phosphorescence quenching). Further, contrary to 30 mins of mechanical ventilation, 6 hrs of mechanical ventilation significantly compromised the diaphragm's ability to increase blood flow during electrically-induced contractions, which resulted in a ~80% reduction in diaphragm oxygen uptake. In contrast, 6 hrs of spontaneous breathing in anesthetized animals did not alter diaphragm blood flow or the ability to augment flow during electrically-induced contractions. These new and important findings reveal that prolonged mechanical ventilation results in a time-dependent decrease in the ability of the diaphragm to augment blood flow to match oxygen demand in response to contractile activity and could be a key contributing factor to difficult weaning. Although additional experiments are required to confirm, it is tempting to speculate that this ventilator-induced decline in diaphragmatic oxygenation could promote a hypoxia-induced generation of reactive oxygen species in diaphragm muscle fibers and contribute to ventilator-induced diaphragmatic atrophy and contractile dysfunction.